Porcine protein and uses thereof

ABSTRACT

The present invention provides a porcine antigen that binds to human xenoreactive antibodies. The porcine antigen differs from the known porcine xenoantigens in that the antigen does not include an αGal epitope. The present invention also provides methods to purify the porcine antigen of the invention, as well as agents that bind to the antigen. The antigen may be used to generate antibodies against the antigen. The antigen is useful for detecting the presence of human xenoreactive antibodies against the antigen in blood and blood compositions, and antibodies against the antigen may be used to detect the presence of the antigen in samples. The invention also provides methods and pharmaceutical compositions for reducing a host rejection response to a porcine xenograft. Finally, a method to treat human blood or blood-derived compositions to reduce the level of human xenoantibodies is disclosed.

The subject application is a continuation of U.S. Ser. No. 09/277,391,filed Mar. 26, 1999, now U.S. Pat. No. 6,245,890 B1, issued Jun. 12,2001, the contents of which are hereby incorporated in their entiretyinto the subject application.

FIELD OF THE INVENTION

The present invention relates to a novel porcine protein that isinvolved in xenograft rejection in humans. Specifically, the presentinvention relates to a porcine protein, found in porcine red bloodcells, that binds to human serum antibodies involved in xenograftrejection, but which does not bind to human serum antibodies against thedominant xenograft rejection antigen, namely, the αGal epitope. Thepresent invention also relates to methods for the detection, isolationand use of the porcine red blood cell protein, as well as methods forthe detection, isolation and use of human serum antibodies that bind tothe protein. Finally, pharmaceutical compositions and methods oftreatment are also provided.

BACKGROUND OF THE INVENTION

Organ transplantation has become the treatment of choice for variousdiseases associated with organ failure (Platt J L (1998) Nature392:11-17; Auchincloss H Jr, and Sachs D H (1998) Ann Rev Immunol16:433-70). However, the supply of organs from organ donors falls farshort of meeting the rapidly increasing demand (Hammer C, Linke R,Wagner F, and Diefenbeck M (1998) Int Arch Allergy Immunol 116:5-21). Inthe United States, only 5% of the patients on various waiting lists fororgan transplants ever receive the appropriate organs. One attractiveapproach to overcoming this shortage is to use animals as a source oforgans for transplantation (Greenstein J L and Sachs D H (1997) NatBiotechnol 15:235-238). The transplantation of organs or cells betweenmembers of different species is called xenotransplantation. Non-humanprimates are the closest biological relatives of human beings;therefore, their organs are most similar to humans, anatomically,physiologically and biochemically. In fact, organs from chimpanzees andbaboons have been shown in clinical studies to exhibit extendedxenograft survival following rather simple immunosuppression procedures(Bailey L L, Nehlsen-Cannarella S L, Concepcion W, and Jolley W B (1985)JAMA 254(23):3321-3329; Starzl T, Marchioro T L, and Peters G (1964)Transplantation 2:752-776). However, the large-scale production of theseendangered non-human primates for the purpose of securing organs fortransplantation is considered unethical and socially unacceptable(Cortesini R (1998) Transplant Proc 30(5):2463-2464). Furthermore, thetransmission of pathogens from primates to humans is well documented,and pathogen-free primates are extremely difficult to raise (Allan J S(1995) Nat Medicine 2(1):18-21). In addition, a significant obstacle tothe widespread adoption of xenotransplantation is the immunologicalincompatibility between nonprimate animals and humans, which results instrong host rejection responses to the xenotransplanted organ.Overcoming these host rejection responses is essential to enablewidespread use of nonprimate animal organs in xenotransplantation.

The first immune barrier to xenograft survival is hyperacute rejection,which may occur within minutes after revascularization of an organ(Rosenberg J C, Hawkins E, and Rector F (1971) Transplantation11(2):151-157). The rejection is induced by the activation of the host(recipient) complement cascade upon the binding of recipientxenoreactive natural antibodies to the xenograft. The major xenograftantigen (or “xenoantigen”) responsible for the hyperacute rejectionresponse has been identified as a carbohydrate epitope,Galα1,3Galβ1,4GlcNAc (referred to herein as “the αGal epitope”). TheαGal epitope forms glycoconjugates on the cell surface of animal organsand cells (Thall A, and Galili U (1990) Biochemist 29: 3959-3965; Good AH, Cooper D K C, Malcolm A J, Ippolito R M, Koren E, Neething F A, Ye Y,Zuhdi N and Lamontagne L R (1992) Transplantation Proceedings24:559-562). The α-Gal epitope is universally present in the animalkingdom, with the exception of humans and Old World monkeys who lack thegalactosyltransferase responsible for the epitope synthesis. Conversely,in normal human serum there are significant amounts of naturallyoccurring anti-αGal antibodies, which constitute approximately 1-3% oftotal IgG molecules and 3-5% of total IgM (Rother R P and Squinto S P(1996) Cell 86:185-188). Recent studies suggest that anti-αGal IgM,rather than IgG, is responsible for the hyperacute rejection responseobserved in organ xenotransplantation (Kroshus T J, Bolman R M III, andDalmasso A P (1996) Transplantation 62:5-12).

The second immunological barrier to xenografts is termed delayed orvascular rejection. Although a detailed mechanism has yet to beelucidated, vascular endothelium cells are considered to be the targetfor immune activation through antibody-dependent cytotoxicity mediatedby NK cells and macrophages (Lawson J H, and Platt J L (1996)Transplantation 63:303-310). In contrast to hyperacute rejection, bothIgG and IgM induce vascular rejection effectively, and complement isapparently not involved. These antibodies may represent xenoreactivenatural antibodies whose specificities have not yet been characterized.Finally, the cellular immune response constitutes the last barrier forxenotransplantation. The mechanism involved is probably similar to thatobserved in allograft rejection, but with more potent responses.

Although immune responses to xenografts are divided into three stages,most research and clinical strategies thus far developed have been aimedonly at the first stage, hyperacute rejection. One approach to reducinghyperacute rejection involved circulating human recipient blood over anαGal immunoadsorbent column to remove anti-αGal antibodies prior totransplantation of the xenograft (Taniguchi S, Neethling F A, KorchaginaE Y, Bovin N, Ye Y, Kobayashi T, Niekrasz M, Li S, Koren E, Oriol R,Cooper D K C (1996) Transplantation 62:1379-1384). However, while theprocedure was successful in reducing the concentration of anti-αGalantibodies in the human blood, the reduction in antibody levels wastransient, and was often followed by a rapid rebound within days. A moreattractive alternative is to alter the αGal epitope on the donor organby expressing or knocking out specific glycosyltransferase/glyco-sidaseactivities in organ donor animals (Osman N, McKenzie I F C, Ostenried K,Ioanou Y A, Desnick R J, and Sandrin M S (1997) Proc Natl Acad Sci94:14677 -14682; Koike C, Kannag, R, Takuma Y, Akutsu F, Hayashi S,Hiraiwa N, Kadomatsu K, Muramatsu T, Yamakawa H, Nagai T, Kobayashi S,Okada H, Nakashima I, Uchida K, Yokoyama I, and Takagi H (1996)Xenotransplantation 3:81-86) (see FIG. 1). A third approach is togenerate transgenic animals that express human complement regulatoryproteins, such as DAF, CD46 and CD59 (Zaidi A, Schmoeckel M, Bhatti F,Waterworth P, Tolan M, Cozzi E, Chavez G, Langford G, Thiru S, WallworkJ, White D, and Friend P (1998) Transplantation 65: 1584-1590). In thisapproach, although binding of xenoreactive antibodies still takes place,the presence of these regulatory proteins may prevent complement-inducedcell lysis. Data from several studies seem to suggest that a combinationof different approaches may be required to efficiently inhibithyperacute rejection associated with xenograft transplantation.

In recent years, a consensus has emerged that the domestic pig mayrepresent a good alternative to nonhuman primates as a donor of organsfor transplantation. Porcine and human organs have similar sizes andcardiac output efficiencies. Pigs are relatively easy and inexpensive toraise in large numbers. Furthermore, pigs can be more easily raised insterilized environments than nonhuman primates, and the use of pigs asorgan donors produces fewer ethical concerns. The greater phylogeneticdistance between pigs and humans means it is less likely that xenograftsof pig organs or cells would impose any realistic risk of transmittingan infectious organism of epidemiological significance to the humanpopulation. However, the immunological and physiological incompatibilitybetween pigs and humans remains a significant obstacle to the widespreaduse of pig organs for transplantation.

Advances in the field of xenotransplantation have brought to light theintriguing prospect of using porcine blood for use in xenotransfusions.Serologically, human blood and pig blood have a number of importantfeatures in common, including a similar hematocrit, blood volume, andnumber of blood groups. Biochemically, human hemoglobin has beenexpressed in pig and functions normally in vivo (Rao M J, Schneider K,Chait B T, Chao T L, Keller H, Anderson S, Manjula B N, Kumar R, andAcharya A S (1994) Artif Cells Blood Substit Immobil Biotechnol22:695-700). However, surface antigens on porcine red blood cells(“pRBCs”) are significantly different from those identified on human redblood cells, and are recognized by antibodies in human serum. Thus,pRBCs would certainly be short lived if injected into human bloodcirculation. Since donor endothelium is not involved, the reactions inhuman immunological responses to pRBCs are probably different from thereactions associated with organ transplantation. One may gain someinsight into the human immune responses to pRBCs from a consideration ofthe fate of mismatched red cells upon allo-transfusion in humans. Thereare two principal mechanisms of in vivo red cell destruction inallo-transfusion (Transfusion Reactions in Applied Blood Group Serology,Chapter 36, 4^(th) Edition (1998) (Eds. Issitt, P D and Anstee D J)Montgomery Scientific Publications, Durham, N.C.). The first mechanism,termed intravascular destruction, is triggered by antibody binding andcomplement activation. Red cells are thus rapidly lysed and releasehemoglobin into the blood stream. IgG molecules may play a major role inintravascular destruction, since many IgM antibodies against bloodgroups are cold-reactive and do not bind efficiently to red cells invivo. The second mechanism for clearing red cells in vivo isextravascular destruction, in which intact red cells are removed fromcirculation primarily by macrophages in the liver and spleen. In thiscase red cells are coated with IgG, but not at a high enoughconcentration to activate the complete complement cascade. Under theseconditions, the complement cascade is usually halted at the C3 stage.

As is the case with other porcine cells thus far studied, pRBCs carrythe αGal epitope on the cell surface, and this epitope accounts for themajor reaction observed with xenoreactive human natural antibodies.However, there are data suggesting that porcine red cells may also carryadditional xenoantigens other than the αGal epitope (MacLaren L, Lee T DG, Anderson D, Nass M, and McAlister V C (1998) Transplant Proc30:2468). The natures of these non-αGal xenoantigens and correspondinghuman xenoreactive antibodies have not been explored.

Identification of these non-αGal epitopes and finding ways to reduce oreliminate host immune rejection responses directed to these epitopes isimportant to fully realize the potential benefits of using porcineorgans in xenotransplantation.

SUMMARY OF THE INVENTION

The present invention is directed to a porcine xenoantigen that does notinclude an αGal epitope. The present invention is also directed toagents which bind to the porcine xenoantigen.

The present invention also provides methods for the isolation of theporcine xenoantigen and for the isolation of human xenoreactiveantibodies that bind to the antigen, as well as kits useful inpracticing these methods.

The present invention further provides methods and pharmaceuticalcompositions for treating a host rejection response to a xenograft whichemploy the isolated porcine xenoantigen, or the agents that bind to theantigen.

The present invention still further provides a method for treating aporcine xenograft that renders it suitable for transplantation into asubject in need of such a xenograft. The method includes treating thexenograft with the porcine xenoantigen or, alternatively, an agent whichbinds the porcine xenoantigen.

A method for removing human antibodies that react with the porcinexenoantigen is also provided by the present invention.

Additional objects of the invention will be apparent from thedescription which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents possible transgenic approaches to eliminating the αGalepitope in animals. The αGal epitope (Galα1,3Galβ1,4GlcNAc), synthesizedby α1,3 galactosyltransferase, is the major antigen responsible forhyperacute rejection responses. Strategies that are being tested toblock the αGal epitope include, but are not limited to, the in vivoexpression of α1,2 fucosyltransferase, sialyl-transferase andα-galactosidase, and the production of α,3 galactosyltransferaseknockout animals.

FIG. 2 represents the chemical structure of the αGal epitope and relatedoligosaccharide structures.

FIG. 3 represents the procedure for measuring human complement-mediatedhemolysis. The procedure is outlined in panel B. Hemolysis (A) isplotted as percent lysis versus volume of human serum added (μl). Lysisis defined as (OD-OD_(o))/(OD₁₀₀−OD_(o)), where OD represents the amountof lysis, as measured by the absorbance at 541 nm, OD_(o) is theabsorbance at 541 nm that is due to spontaneous lysis in the absence ofany additions, and OD₁₀₀ is the absorbance at 541 nm that is due to 100%lysis of red cells under the experimental conditions.

FIG. 4 shows a graph depicting the effect of the αGal epitopetrisaccharide on complement-mediated hemolysis. Curve 1: human serumpre-incubated with the trisaccharide, Galα1,3Galβ1,4GlcNAc, at 4° C. for1 hr, followed by a hemolysis assay as described in FIG. 3, panel B.Curve 2: hemolysis induced by serum alone without the inhibitor. Curve3: hemolysis induced by heated-inactivated serum as a negative control.

FIG. 5 shows the time-course for complement-induced hemolysis in thepresence or absence of the trisaccharide of FIG. 4. The hemolysis wasmonitored up to 4 hr. The data were treated by regression curve fitting(Sigmoid Hill equation).

FIG. 6 represents a flow diagram showing the fractionation procedure ofnon-anti-αGal xenoreactive human antibodies from pooled blood type ABserum.

FIG. 7 represents the effect of the trisaccharide of FIG. 4 on thebinding of fractions S1 and S2 to pRBCs. 40 μl of the trisaccharide (2mg/ml) was added to 2 μl of S1 or S2 plus 38 μl of PBS (finalconcentration of trisaccharide 1 mg/ml), followed by incubation at 4° C.overnight. pRBCs were then added and flow cytometry analysis performed,using anti-human IgG (H+L) conjugated with phycoerythrin (PE) as thesecondary antibody. Sample 1: negative control lacking the primary andsecondary antibodies. Sample 2: negative control containing thesecondary antibody only. Samples 3 and 4: fraction S1 without and withthe trisaccharide of FIG. 4, respectively. Samples 5 and 6: fraction S2without and with the trisaccharide of FIG. 4, respectively.

FIG. 8 represents the effect of concentration of the trisaccharide ofFIG. 4 on the binding of S1 to pRBCs. Fraction S1 was incubated withvarious amounts of the trisaccharide under the same conditions describedin FIG. 4. The binding of IgG (curve 1) and IgM (curve 2) molecules fromfraction S1 was monitored by flow cytometry, using anti-human IgG andanti-human IgM, respectively. The data from the flow cytometry analyseswere plotted as mean fluorescent intensity (MFI) versus the finalconcentration of trisaccharide, and the curves were generated bynonlinear curve fitting.

FIG. 9 represents the effect of the trisaccharide of FIG. 4 on thebinding of anti-αGal and non-αGal antibodies to pRBCs. The controlsamples (1 and 2) were as described for FIG. 7. (A): anti-αGal antibodyincubated in the absence (curve 3) or presence (curve 4) of thetrisaccharide (1 mg/ml). (B): Non-αGal antibodies incubated in theabsence (curve 3) or presence (curve 4) of the trisaccharide (1 mg/ml).

FIG. 10 represents the detection of pRBC-bound IgG and IgM by fractionS2 and the non-anti-αGal antibody fraction. PE-conjugated anti-human IgG(or IgM) antibodies were used as the secondary antibodies. (A):pRBC-bound IgG (2) and IgM (3) molecules incubated with fraction S2.(B): pRBC-bound IgG (4) and IgM (5) incubated with non-anti-αGalantibodies. Curve 1: negative control lacking the primary and secondaryantibodies.

FIG. 11 represents the effect of ssDNA and thyroglobulin on the bindingof S2 to pRBCs. The S2 fraction was pre-incubated with ssDNA (2),thyroglobulin (3), or with no inhibitor (3) at 4° C. for 3 hr prior toflow cytometry analyses. Curve 1: negative control lacking the primaryand secondary antibodies.

FIG. 12 shows a flow chart of the procedure employed in the isolation ofporcine xenoantigens that bind to human non-αGal antibodies.

FIG. 13 represents the flow cytometry analyses of non-anti-αGal IgG andIgM bound to pRBCs during the isolation of xenoantigens. Samples wereincubated at 37° for 1 hr in the absence (2) or presence (3) of pRBCs,followed by incubation at 4° overnight. Thereafter, the samples wereanalyzed by flow cytometry. (A) anti-human IgM used as the secondaryantibody. (B) anti-human IgG used as the secondary antibody. Curve 1:negative control lacking the primary and secondary antibodies.

FIG. 14 represents SDS-polyacrylamide gel electrophoresis (SDS-PAGE)analysis of pRBC membrane proteins isolated by the procedure of FIG. 12.(A) Samples from different steps of purification were separated bySDS-PAGE, and visualized by silver staining. Lane 1: red cell membranes(3 μl). Lane 2: unbound fraction from the anti-human IgG and anti-humanIgM columns (10 μl). Lane 3: samples loaded onto the anti-human IgGcolumn (10 μl). Lane 4: eluate from the anti-IgM column (15 μl). Lane 5:eluate from the anti-IgG column (15 μl). (M): Molecular weight standardsexpressed in kDa. (B) Concentrated eluate from the anti-human IgG columnwas separated by SDS-PAGE (1), then transferred to a PVDF membrane andvisualized by Coomassie blue staining. Protein bands marked H and Lcorresponded to immunoglobulin heavy and light chains, respectively. The45 kDa band (arrow) was excised for direct N-terminal sequencing. (M):Pre-stained protein molecular weight markers, expressed in kDa.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a purified porcine xenoantigen, found onporcine red blood cells, which binds to a xenoreactive human antibodyand which does not include an αGal epitope. As used herein, axenoantigen is an antigen which is found in or on cells of a nonhumananimal and which is not found in or on human cells. A xenoreactive humanantibody refers to a human antibody that binds specifically to axenoantigen. The purified antigen is preferably a protein, and furtherpreferably is a protein having a molecular weight of about 45 kDa, asmeasured by SDS polyacrylamide gel electrophoresis. The purified antigenis also preferably a protein comprising the amino acid sequenceAsp-Val-Xaa-Pro-Val-Glu-Ser-Val-Xaa (SEQ ID NO:1), wherein Xaa refers toany of the twenty amino acids which occur naturally in mammals. Theamino acid sequence of SEQ ID NO: 1 may be an amino-terminal sequence.The antigen may also be a polysaccharide or a lipid, or any combinationof a protein, a polysaccharide, or a lipid, so long as the antigen doesnot include an αGal epitope. The antigen of the present invention may beisolated from porcine red blood cells or recombinantly produced. It isalso within the confines of the present invention that the antigenincludes similar or identical antigens from other porcine tissues whichbind to a xenoreactive human antibody and which do not include an αGalepitope.

The present invention also provides a purified agent which binds to apurified porcine xenoantigen. The antigen is any that does not includean αGal epitope. More specifically, the porcine xenoantigen is found onporcine red blood cells. Preferably, the agent is any molecule thatspecifically binds to a purified porcine xenoantigen and inactivates orblocks the binding of the antigen to a xenoreactive human antibody. Theagent may be in the form of an antibody, an Fab fragment, an F(ab′)₂fragment, an Fv antibody, a drug, a peptide, a protein, a nucleic acid,a lipid, a polysaccharide, or any combination thereof. An Fab fragmentis a univalent antigen-binding fragment of an antibody which is producedby papain digestion. An F(ab′)₂ fragment is a divalent antigen-bindingfragment of an antibody which is produced by pepsin digestion. An Fvantibody is a hybrid antibody molecule usually produced by phage displaymethods. Antibodies useful in the present invention may be eithermonoclonal antibodies or polyclonal antibodies. Antibodies may beproduced by any of a number of methods well-known to those in the art.Drugs include small organic or inorganic molecules. Where the agent is apolysaccharide, any polysaccharide can be used that is effective to bindto a purified porcine xenoantigen and block the binding of the antigento a xenoreactive human antibody, so long as the polysaccharide does notcontain an αGal epitope.

The present invention also provides a method for the isolation ofnon-αGal porcine xenoantigens from porcine tissues or cells. The methodcomprises the following steps: (a) contacting human sera with an α-Galepitope-containing molecule, whereby an antibody in the human sera bindsto the α-Gal epitope-containing molecule to form a complex; (b) removingthe complex from the human sera to produce an anti-αGalantibody-depleted sera; (c) contacting the anti-αGal antibody-depletedsera with porcine tissue or cells, whereby at least one humanxenoreactive antibody present in the anti-αGal antibody-depleted serabinds to an antigen present on the porcine tissue or cells; (d)isolating the tissue or cells bound to the xenoreactive antibody; (e)separating the human xenoreactive antibody bound to the antigen from thetissue or cells; and (f) eluting the antigen from the human xenoreactiveantibody.

The αGal epitope-containing molecule of the method described abovepreferably includes the trisaccharide Galα1,3Galβ1,4GlcNAc, and isfurther preferably attached to a solid support, such as an insolubleorganic polymer in the form of a bead, gel or plate. Examples ofsuitable solid supports include, without limitation, dextran, agarose,cellulose, polystyrene, polyacrylamide, or other insoluble organicpolymers. The trisaccharide attached to the solid support may be used asa slurry, or in the form of a column, cartridge, or plate. The αGalepitope may further be attached to the solid support through a spacermolecule if desired.

The αGal epitope-containing molecule binds to an anti-αGal antibody inthe serum to produce a complex, e.g., an antigen/antibody complex, andan anti-αGal antibody-depleted serum. After binding, the complex may beseparated from the serum by methods well known to those in the art. Forexample, if the trisaccharide attached to the solid support is in theform of a column or a cartridge, the serum may be passed through thecolumn or cartridge and the resulting serum extract treated as theanti-αGal antibody-depleted serum. If the trisaccharide is attached tothe solid support in the form of a plate, or is attached directly to aplate, the serum may be passed over the surface of the plate by panningand then removed and thereafter treated as the anti-αGalantibody-depleted serum. If the trisaccharide attached to the solidsupport is in the form of a slurry, the complex may be separated fromthe serum by filtration, or by sedimenting or centrifuging the slurry,followed by decanting to separate the liquid phase from thesolid-containing phase. If the trisaccharide is not bound to a solidsupport, the binding may also be performed under conditions whereby thecomplex is rendered insoluble upon formation. Thereafter, the complexmay be removed from the serum by any appropriate method, including butnot limited to sedimentation, centrifugation or filtration.

After the αGal-antibody depleted serum is obtained, it is contacted withporcine tissue or cells. The porcine tissue or cells may be any tissueor cells that bears a non-αGal epitope xenoantigen. Suitable tissues orcells may include endothelial cells, liver, heart, brain, kidney,intestine, pancreas, lung, skin, bone marrow, blood, red blood cells,and the like, and preferably porcine red blood cells or porcine redblood cell membranes, or any derivatives thereof. The tissue or cellsare contacted with the αGal-antibody depleted serum under conditionsthat promote binding of the antigen to a non-anti-αGal humanxenoreactive antibody in the αGal-antibody depleted serum. Theseappropriate bind conditions (e.g. temperature, pH and saltconcentration) are readily determinable by the skilled artisan. Aftercontacting the tissue or cells with the αGal-antibody depleted serum fora period of time sufficient to bind the antibody, the tissue or cells isthereafter separated from the serum by any appropriate means. Thesemeans may include, but are not limited to, sedimentation,centrifugation, or filtration. Alternatively, the tissue or cells may bebound to a solid support in the form of a column, cartridge or plate,and the serum separated from the tissue or cells by passage through thecolumn or cartridge or by panning over the plate.

Thereafter, the tissue or cells are contacted with a purified antibodythat binds selectively to the non-anti-αGal human xenoreactive antibodybound to the antigen on the tissue. The purified antibody may be ananti-human IgG antibody, an anti-human IgM antibody, or a combination ofthese. These antibodies are widely available through commercialsuppliers and are well known to those in the art. Again, the purifiedantibody is contacted with the tissue or cells under conditions(temperature, pH, and salt concentration) that promote binding of thepurified antibody to the human xenoreactive antibody bound to theantigen on the tissue or cells. Thereafter, the tissue or cells areseparated from the purified antibody, which is bound to the humanxenoreactive antibody, which in turn is bound to the antigen. Theseparation is performed under conditions that permit the ternary complexof purified antibody-xenoreactive antibody-antigen to remain intact, yetbe separated from the remainder of the tissue or cells and othercomponents of the mixture. This may require treatment of the tissue orcells in any of a number of ways, such as, without limitation, physicalor enzymatic dissociation of the tissue or solubilization by detergents.This separation is possible in view of the strong interaction betweenantibodies and their antigens. However, any procedures used to separatethe antigen from the tissue or cells preferably does not also result inthe dissociation of the components of the ternary complex.

Thereafter, the antigen is eluted from the ternary complex by anyappropriate method. Examples include heat elution (i.e., 57° C. for 5min) or elution using acid or base. The eluted antigen may thereafter befurther purified, if required, by appropriate methods well known tothose in the art.

The present invention also provides a method for isolating a humanxenoreactive antibody which binds to a porcine xenoantigen that does notinclude an αGal epitope. The method comprises the steps of: (a)contacting human sera with an α-Gal epitope-containing molecule, wherebyan antibody in the human sera binds to the α-Gal epitope-containingmolecule to form a complex; (b) removing the complex from the human serato produce an anti-αGal antibody-depleted sera; (c) contacting theanti-αGal antibody-depleted sera with porcine tissue or cells, wherebyat least one human xenoreactive antibody present in the anti-αGalantibody-depleted sera binds to an antigen present on the porcine tissueor cells; (d) isolating the tissue or cells bound to the xenoreactiveantibody; (e) separating the human xenoreactive antibody bound to theantigen from the tissue or cells; and (f) eluting the human xenoreactiveantibody from the antigen.

It will be appreciated that the method for isolation of the agentoutlined above is very similar to that previously discussed forisolating the antigen, with the exception of the final step (step (f)).The steps for eluting the human xenoreactive antibody from the purifiedantibody implicate the same concerns as for the elution of the antigen.Therefore, the human xenoreactive antibody may be purified from theanti-human antibody and the porcine antigen by any appropriate means, asoutlined above for the antigen.

The present invention also provides a method for detecting the presenceof a porcine red blood cell antigen in a sample. The method may beperformed by contacting a sample with an agent which binds to a porcineantigen under conditions permitting the antigen, if present in thesample, to bind to the agent to form a complex, and thereafter detectingthe presence of the complex. The antigen is one that does not include anαGal epitope. The agent may be any of an antibody, an Fab fragment, anF(ab′)₂ fragment, an Fv antibody, a drug, a peptide, a protein, anucleic acid, a lipid, a polysaccharide, or any combination thereof. Theagent is preferably an antibody, and more preferably a monoclonalantibody, directed against the antigen. Where the agent is an antibody,an Fab fragment, an F(ab′)₂ fragment, or an Fv antibody, the method maybe performed as an ELISA assay, a Western blot, or as any otherimmunostaining method employing an antigen-antibody interaction. Thepresent invention also contemplates the provision of a kit forperforming the method of detecting the presence of a porcine antigen ina sample, as described above. The kit would include a container, anagent which binds to a porcine antigen to form a complex, and a reagentor reagents capable of detecting the resulting complex. As noted above,the antigen is one that binds to a human xenoreactive human antibody,and which does not include an αGal epitope. The reagent or reagentscapable of detecting the complex are preferably secondary antibodiesthat bind selectively to one or the other of the antigen and the agent,preferably the agent, and which are further linked, either through acovalent linkage or by a noncovalent linkage, to a reporter molecule,including but not limited to an enzyme, a fluorescent molecule, aradioactive molecule, or a light-emitting molecule.

The present invention also provides a method for detecting the presence,in a sample, of a xenoreactive human antibody that binds to a porcineantigen. The method may be performed by contacting a sample with aporcine antigen that binds to a xenoreactive human antibody to form anantigen-antibody complex, and thereafter detecting the presence of theantigen-antibody complex. The porcine antigen is one that does notinclude an αGal epitope. The method may be performed as an ELISA assay,a Western blot, or as any other immunostaining method employing anantigen-antibody interaction. The present invention also contemplatesthe provision of a kit for performing the method of detecting thepresence of a xenoreactive human antibody that binds to a porcineantigen in a sample, as described above. The kit would include acontainer, a porcine antigen, and a reagent or reagents capable ofdetecting the resulting antigen-antibody complex. As noted above, theporcine antigen is one that binds to a human xenoreactive humanantibody, and which does not include an αGal epitope. The reagent orreagents capable of detecting the complex are preferably secondaryantibodies that bind selectively to one or the other of the antigen andthe agent, preferably the agent, and which are further linked, eitherthrough a covalent linkage or by a noncovalent linkage, to a reportermolecule, including but not limited to an enzyme, a fluorescentmolecule, a radioactive molecule, a light-emitting molecule, and otherknown reporter molecules.

The present invention also provides a method of treating a hostrejection response to a xenograft in a subject in need of such treatmentwhich comprises administering either a purified antigen that binds to ahuman xenoreactive antibody, or an agent which binds to the purifiedagent that binds to a human xenoreactive antibody. In either case, theantigen or agent is administered in an amount which is effective toreduce the host rejection response in the subject. That is, the amountadministered is an amount effective to reduce one or more of thehyperacute rejection, the delayed or vascular rejection, and thecellular immune response associated with xenotransplantation. Thisamount is readily determinable by one skilled in the art, and may varyamong patients. Administration may be by any appropriate method,including but not limited to oral, sublingual, parenteral, ortransdermal administration, or administration by suppository, andpreferably parenteral administration.

Furthermore, a pharmaceutical composition for treating a host rejectionresponse to a xenograft comprises either a purified antigen that bindsto a human xenoreactive antibody, or an agent which binds to thepurified agent that binds to a human xenoreactive antibody. In eithercase, the antigen or agent is provided in an amount which is effectiveto reduce the host rejection response in the subject. The pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. Examples of suitablepharmaceutical carriers include lactose, sucrose, starch, talc,magnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate, gum arabic, powders,saline, and water, among others. The formulations may conveniently bepresented in unit dosage and may be prepared by methods well-known inthe pharmaceutical art, by bringing the active compound into associationwith a carrier or diluent, as a suspension or solution, and optionallyadding one or more accessory ingredients, e.g. buffers, flavoringagents, surface active agents, and the like. The choice of carrier willdepend upon the route of administration.

For oral and sublingual administration, the formulation may be presentedas capsules, tablets, powders, granules or a suspension, withconventional additives such as lactose, mannitol, corn starch or potatostarch; with binders such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators suchas corn starch, potato starch or sodium carboxymethylcellulose; and withlubricants such as talc or magnesium stearate.

For parenteral administration, the antigen or agent may combined with asterile aqueous solution which is preferably isotonic with the blood ofthe recipient. Such formulations may be prepared by dissolving a solidactive ingredient in water containing physiologically compatiblesubstances such as sodium chloride, glycine, and the like, and having abuffered pH compatible with physiological conditions to produce anaqueous solution, and rendering the solution sterile. The formulationsmay be present in unit or multi-dose containers such as sealed ampoulesor vials.

For transdermal administration, the antigen or agent may be combinedwith skin penetration enhancers such as propylene glycol, polyethyleneglycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and thelike, which increase the permeability of the skin to the compounds, andpermit the compounds to penetrate through the skin and into thebloodstream. The antigen or agent also may be combined additionally witha polymeric substance such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which can be dissolved in a solvent such asmethylene chloride, evaporated to the desired viscosity, and thenapplied to backing material to provide a patch.

For administration by suppository, the antigen or agent may be combinedwith any appropriate base to form a mass that is solid at roomtemperature but dissolves at body temperature. The base may include,without limitation, cocoa butter, glycerinated gelatin, hydrogenatedvegetable oils, polyethylene glycols of various molecular weights, fattyacid esters of polyethylene glycols, and the like.

The present invention also provides a method of treating a porcinexenograft to render the xenograft suitable for transplantation,comprising the step of contacting the xenograft with an agent that bindsa porcine xenoantigen in an amount effective to reduce a host rejectionresponse in a subject receiving the xenograft. The porcine xenoantigenis one that does not include an αGal epitope. The present invention alsoprovides a xenograft that has been treated according to the methodoutlined above. The xenograft may include, without limitation, blood, ablood product, a red blood cell composition, endothelial cells, bonemarrow, a liver, a kidney, a lung, or a heart, or any combinationthereof. The amount of the agent administered is an amount effective toreduce the host rejection response in the subject, by blocking theinteraction of the porcine xenoantigen on the xenograft with humanxenoreactive antibodies. The amount administered is further an amounteffective to reduce one or more of the hyperacute rejection, the delayedor vascular rejection, and the cellular immune response associated withxenotransplantation. This amount is readily determinable by one skilledin the art, and may vary among patients.

The present invention also provides a method of removing humanxenoreactive antibodies from blood or a blood-derived composition,comprising the steps of contacting the blood or blood-derivedcomposition with an antigen which binds to a human xenoreactive antibodyto form an antigen-antibody complex, and removing the resultingantigen-antibody complex from the blood or blood-derived composition.The present invention also provides a blood or blood-derived compositionthat has been treated by the above-described method. As noted above, theantigen does not include an αGal epitope. The antigen may be attached toa solid support, such as an insoluble organic polymer in the form of abead, gel or plate. Examples include, without limitation, dextran,agarose, cellulose, polystyrene, polyacrylamide, or other insolubleorganic polymers. The antigen attached to the solid support may be usedas a slurry, or in the form of a column, cartridge or plate. The antigenmay further be attached to the solid support through a spacer moleculeif desired.

The antigen binds to xenoreactive antibodies in the blood orblood-derived composition to produce a complex, e.g., anantigen/antibody complex. After binding, the complex may be removed fromthe blood or blood-derived composition by methods well known to those inthe art. For example, if the antigen attached to the solid support is inthe form of a column or a cartridge, the blood or blood-derivedcomposition may be passed through the column or cartridge. If theantigen is attached to a solid support in the form of a plate, orattached directly to a plates the blood or blood-derived composition maybe passed over the surface of the plate by panning and then removed. Ifthe antigen attached to the solid support is in the form of a slurry,the complex may be separated from the blood or blood-derived compositionby filtration, or by sedimenting or centrifuging the slurry, followed bydecanting to separate the liquid phase from the solid-containing phase.

The present invention is further described in the following ExperimentalDetails section which is set forth to aid in the understanding of theinvention, and should not be construed to limit in any way the scope ofthe invention as defined in the claims which follow thereafter.

Experimental Details

1. pRBC Agglutination With Human Serum And Antibodies Against HumanBlood Group Antigens

At least 16 blood groups on pRBCs have been serologically identified(Erythrocyte antigens: the immune responses to red cells as an exampleof type II reactions in An Introduction to Veterinary Immunology (1982)pp. 276-283). Chromosome loci for 14 of these blood groups have beendetermined. However, their biochemical properties have not yet been wellcharacterized. Even less is known in terms of their correlation withblood group antigens identified on human red blood cells. Toward thatend, we carried out agglutination test using pRBCs and antibodiesagainst various human blood group antigens. As shown in Table 1, pRBCsmay carry type A or type B antigen or neither. The type A carbohydratechain has been previously isolated from pRBCs and is recognized byeither polyclonal or monoclonal anti-A in our studies. On the otherhand, monoclonal anti-B antibody selectively agglutinates type B pRBCswhereas polyclonal anti-B reacts with all the samples tested. Thesimplest explanation is that the polyclonal, but not the monoclonal,antibody may contain a subset that interacts with the ubiquitous αGalepitope (linear type B structure). Furthermore, although the αGalepitope is present in all porcine blood samples its level of expressionon the cell surface varies significantly (MacLaren L, Lee T D G,Anderson D, Nass M, and McAlister V C (1998) Transplant Proc 30:2468).

TABLE 1 Antibody Strength Antibody Strength polyclonal anti-A 4 +/−anti-Fy 0 monoclonal anti-A 2 +/− anti-P 0 polyclonal anti-B 4 + anti-P₁0 monoclonal anti-B 1 +/− anti-M 0 anti-A,B 4 + anti-N 0 monoclonalanti-H 0 anti-K 0 monoclonal anti-D 0 anti-k 0 anti-C 0 anti-S 0 anti-c0 anti-s 0 anti-E 0 anti-I 3 + anti-e 0 anti-Le^(b) 0

The only other anti-human blood group antibody that reacts with pRBCs isanti-I. Of all the samples tested, the agglutination strength for anti-Iis consistently between 2+ to 3+, suggesting that the I antigen(Galβ1,4GlcNAc) may exist on all red cells regardless of their otherblood groups (e.g. type A or B). Thus, structurally, the I antigen maybe considered a precursor for the type A, B and the αGal epitopes (FIG.2).

It is not surprising that none of the antibodies against human proteinantigens reacted with pRBCs in hemagglutination assays. Due to theirphylogenetic distance, human protein antigens are most likely to bequite different in terms of antigenicity from their counterparts inpRBCs, if such counterparts exist at all. Rh and Duffy protein are twomajor protein antigens on human red cells, but polyclonal antibodiesagainst these two proteins failed to crossreact with their counterpartsin mouse (Apoil P, and Blancher A (1999) Immunogenetics 49:15-25; Luo H,Chaudhuri A, Johnson K R, Neote K, Zbrzezna V, He Y, and Pogo A O (1997)Genome Res 7(9):932-41). It is thus very interesting to determinewhether any protein antigens on porcine red blood cells are involved inthe binding of xenoreactive natural antibodies in human serum.

When normal human serum from type AB individuals was used in ahemagglutination assay with pRBCs, we observed agglutination strengthsof between 2+ and 3+ with 1 μl of serum in 120 μl reaction. Since thetype AB serum contains neither anti-A nor anti-B antibodies, reactionsinvolving type A or B antigens on pRBCs can thus be excluded. It hasbeen reported that anti-αGal is the major xenoreactive antibody in humanserum and is largely responsible for hemagglutination in vitro. Weprepared an anti-αGal depleted antibody fraction by using a specificligand affinity column, as described below. This fraction produced anagglutination strength of 2+ in the hemagglutination test with pRBCs andwas not affected by pre-incubation with trisaccharideGalα1,3Galβ1,4GlcNAc at a concentration of 1 mg/ml. This is the firstindication that pRBCs are recognized by xenoreactive antibodies otherthan anti-αGal in human serum.

2. Human Complement-Mediated pRBC Hemolysis

In blood transfusions between humans, mismatched red cells undergointravascular destruction by the same mechanism as complement-inducedhemolysis observed in vitro. To shed light on how the binding of humanxenoreactive antibodies to pRBCs triggers the complement cascade andleads to hemolysis, we carried out in vitro complement-induced hemolysisreactions. Human serum containing all complement components waspurchased from Sigma. Since this serum was derived from type Bindividuals, as determined by hemagglutination assays, we used onlynon-type A pRBCs in the complement assays to exclude the binding ofanti-A antibody present in the serum.

The human serum (between 3-18 μl) was mixed with 40 μl of 5% pRBCs(about 2×10⁷ cells) in a total volume of 210 μl. After incubating at 37°C. for 1 hr with constant rotation, the remaining intact cells wereremoved from the reaction by centrifugation. Hemoglobin moleculesreleased from the lysed cells into the supernatant were quantitated bymeasuring the absorbance at 541 nm. As shown in FIG. 3, a plot of pRBClysis (%) versus the volume of serum added (μl) resulted in a typicalS-shaped curve with L₅₀=9 (L₅₀ is defined as the amount of serumrequired to induce 50% of hemolysis). Our data indicate that the bindingof xenoreactive natural antibodies in human serum to pRBCs triggershuman complement-mediated hemolysis in vitro.

In order to assess the contribution of anti-αGal and non-anti-αGalantibodies to the observed lysis, we incubated the human serum withpRBCs in the presence of the αGal epitope trisaccharide (finalconcentration 1 mg/ml) at 4° C. for 1 hr prior to the complement assay.Anti-αGal antibody in normal human serum can be completely inhibited bythe trisaccharide at a concentration of 1 mg/ml, as determined by flowcytometry analysis (see below). As shown in FIG. 4, addition of thetrisaccharide inhibitor significantly reduced the pRBC lysis. Comparedto the control (without inhibitor), 1 mg/ml of trisaccharide blockedapproximately 60% of hemolysis, suggesting that the remaining 40% oflysis may result from the binding of non-anti-αGal xenoantibodiespresent in human serum. As a negative control, human serum that waspre-incubated at 57° C. for 15 min lost complement activity and causedessentially no hemolysis, as shown in FIG. 4. The heat inactivation stepdid not affect the binding of xenoreactive antibodies to pRBCs, asdetermined by flow cytometry analysis (data not shown).

We also measured the time course for complement-mediated hemolysis ofpRBCs (FIG. 5). Cell lysis proceeded rapidly in the first hour ofincubation at 37° C., and then reached a plateau. This was true both inthe presence and absence of 1 mg/ml trisaccharide. In this experiment,we observed approximately 50% inhibition of cell lysis by thetrisaccharide. Another approach to elucidating the role of non-anti-αGalxenoantibodies in hemolysis is to reconstitute antibody-triggered,complement-mediated lysis by adding a non-anti-αGal antibody fraction toantibody-depleted human serum. Antibody-depleted human serum wasprepared by passage over a protein A-resin column. However, numerousattempts failed to generate an antibody-depleted serum with fullcomplement activity. This is presumably due to the loss of somecomplement elements during the antibody absorption by the proteinA-conjugated resin.

3. Fractionation of Xenoreactive Human Natural Antibodies

Pooled human sera from blood type AB individuals were used as thestarting material for xenoreactive antibody fractionation (FIG. 6).After incubation at 56° C. for 15 min to inactivate complement, thehuman serum was subjected to ammonium sulfate precipitation. Proteinsprecipitated between 30% to 50% of (NH₄)₂SO₄ saturation werepredominantly immunoglobulin and designated as fraction S1.

In order to separate anti-αGal antibodies from the S1 fraction, weprepared an affinity column using the trisaccharide as a ligand. A resinwas constructed, using biotinylatedpoly[N-(2-hydroxyethyl)-acrylamide]Galα1,3Galβ1,4GlcNAc (Syntesome,Munich, Germany) and streptavidin-agarose (Pierce, Rockford, Ill., USA).The resulting affinity resin had a capacity of approximately 0.1 nmoleof trisaccharide per ml of resin. Based on reported levels of anti-αGalantibodies in normal human serum (1-3% of total IgG and 3-5% of totalIgM), we added the S1 fraction to the affinity resin at a ratio of lessthan 50 ml of serum per ml of the resin to avoid saturating the column.After mixing for 3 hr at room temperature, the unbound fraction wasseparated and designated as fraction S2. The antibody bound resin wasthen washed extensively with phosphate buffered saline (PBS). Theanti-αGal antibody was finally dissociated from the resin by incubatingat 57° C. for 5 min.

To further isolate antibodies that recognize xenoantigens other than theαGal epitope, we used pRBCs as a specific immunoadsorbent. Afterincubating the S2 fraction with pRBCs for 1 hr at room temperature, theunbound fraction was collected by centrifugation and designated as thepRBC non-reactive fraction (S3). The bound antibodies (non-αGalantibodies) was then recovered from the pRBCs, using either a cold-acidmethod or a heat elusion method.

4. Characterization of Xenoreactive Antibodies by Flow CytometryAnalysis

Various fractions derived from human serum were analyzed by flowcytometry. We first examined the binding of S1 and S2 to pRBCs in thepresence or absence of the trisaccharide inhibitor Galα1,3Galβ1,4GlcNAc.As shown in FIG. 7, the binding of S1 is substantially inhibited by 1mg/ml (1.7 mM) of the trisaccharide, resulting in a drop in MFI from 322(sample 3) to 45 (sample 4). On the other hand, the binding of S2 topRBCs was not significantly affected by the trisaccharide (sample 5 and6). This suggests that the procedure used for the fractionation of humanserum effectively generated an anti-αGal-depleted fraction (S2) withoutcontaminating anti-αGal activity. Furthermore, the results indicate thathuman xenoreactive antibodies other than the well-characterizedanti-αGal antibody recognize pRBCs. In addition, since we used the samevolume (2 μl) of S1 and S2 in the assay, the fact that samples 4, 5 and6 fell into approximately the same position on the histograph suggeststhat the inhibition by free trisaccharide achieved the identical effectas depletion by immobilized trisaccharide.

To demonstrate the inhibitory effect of the trisaccharide on IgG or IgMbinding to pRBCs, flow cytometry analysis was applied. As shown in FIG.8, the trisaccharide seemed to inhibit IgG binding more efficiently thanIgM binding. In a separate experiment, the S1 fraction was pre-incubatedwith the trisaccharide at a concentration up to 10 mM. While there was aslight further reduction in MFI for IgM binding, the binding of IgGremained essentially the same when the inhibitor concentration wasgreater than 1 mM (data not shown). Nevertheless, the binding of bothIgG and IgM was still detectable in the presence of 10 mM trisaccharide,strongly suggesting that non-anti-αGal antibodies play a role in bindingto pRBCs.

To directly detect non-anti-αGal antibodies from the S2 fraction, weisolated these antibodies by using a pRBC absorption/elution method. Theeluted fraction (non-αGal antibodies) was then measured for its bindingto pRBCs in the presence or absence of the trisaccharide. As shown inFIG. 9, while the binding of anti-αGal antibodies was completely blockedby 1 mg/ml of the trisaccharide (FIG. 9A), the inhibitor had essentiallyno effect on the binding of non-anti-αGal antibodies (FIG. 9B). The dataclearly suggest that these non-anti-αGal antibodies are naturallypresent in human serum and interact with certain antigens other than theαGal epitope on pRBCs.

To distinguish between the pRBC-bound IgG and IgM molecules in the S2and non-αGal fractions, we applied PE-conjugated anti-human IgG (γchain) or anti-human IgM (μchain) as the secondary antibody in flowcytometry analysis. As shown in FIG. 10, both types of immunoglobulinare involved in binding to non-αGal epitopes on pRBCs. However, therelative amounts of IgG and IgM in these fractions are difficult toestimate from the graph, since the MFI value is affected by the amountof antibodies used.

To determine whether the xenoreactive antibodies that we characterizedin the binding of pRBCs are polyreactive, we tested the effect of ssDNAand thyroglobulin on binding of antibodies by hemagglutination assaysand flow cytometry analyses (Turman M A, Casali P, Notkins A L, Bach FH, and Platt J L (1991) Transplantation 52:710-717). To ensure that thexenoreactive antibodies were not present in excess, we titrated theamount of antibodies used in the hemagglutination assays and chose thedilution that produced an intermediate strength (2+ or 3+) forinhibition. Fraction S1 or S2 was pre-incubated with ssDNA orthyroglobulin (final concentration 1 mg/ml) at 4° C. for 3 hr beforeadding pRBCs for the hemagglutination assay. As shown in Table 2,neither ssDNA nor thyroglobulin showed any inhibitory effect on S1- orS2-induced hemagglutination. To confirm these results, we assessed thebinding of xenoreactive antibodies to pRBCs in the presence or absenceof ssDNA (or thyroglobulin) by flow cytometry analysis. As shown in FIG.11, the binding was little affected, if any, by pre-incubation withssDNA (or thyroglobulin) at 0.01, 0.1 or 1.0 mg/ml (only the lastconcentration was shown in FIG. 11). Our data suggest that the observedbinding of antibodies to pRBCs is not polyreactive in nature.

TABLE 2 Strength PBS ssDNA (1 mg/ml) thyroglobulin (1 mg/ml) S1 8 μl 4 +4 μl 3 + 3 + 3 + 2 μl 1 + S2 8 μl 4 + 4 μl 2 + 2 + 2 + 2 μl 1 +

5. Isolation of Xenoantigens Recognized by Non-Anti-αGal Antibodies

Although anti-αgal antibody represents the major xenoreactive naturalantibody, non-anti-αGal antibodies in human serum have also beensuggested to be involved in hyperacute rejection and possibly vascularrejection (Maccbiarini P, Oriol R, Azimzadeh A, deMontpreville V, RievenR, Bevin N, Mizmanian M, and Maartevelle P (1998) J Thoracic andCardiovascular Surgery 116:831-843). However, non-αGal antigens that arerecognized by xenoreactive human natural antibodies have not beenidentified and characterized. Therefore, we isolated non-αGal antigensfrom pRBCs by using an anti-αGal-depleted serum fraction (S2).

The procedure for the isolation of non-αGal xenoantigens from pRBCs (150ml of packed cells) is outlined in FIG. 12. To ensure the completebinding of non-anti-αGal antibodies under the conditions applied, thesamples from before and after pRBC absorption were analyzed by flowcytometry using both anti-human IgG and anti-human IgM as secondaryantibody. As shown-in FIG. 13, non-anti-αGal antibodies (both IgG andIgM) were completely absorbed by pRBCs. The final step of thepurification involved two affinity columns. The membrane lysatecontaining immune complexes were first loaded onto the anti-human IgGcolumn. The unbound fraction from the column was then loaded to a secondcolumn (anti-human IgM). In this fashion, we were able to separateIgG-bound and IgM-bound xenoantigens.

Protein samples from different stages of the purification were subjectedto SDS-PAGE analysis. As shown in FIG. 14(A), the eluate (lane 6) fromthe anti-human IgG column consisted of a major band at approximately 45kDa, and a couple of minor bands in addition to immunoglobulin heavychain (53 kDa) and light chain (23 kDa). However, the eluate (lane 5)from the anti-human IgM column was too low in protein quantity to carryout further characterization. The 45 kDa protein may represent the majorprotein antigen on pRBCs that is recognized by non-anti-αGal antibodies(IgG) in human serum. In order to obtain the N-terminal sequence of thisnovel protein, the eluate from the anti-human IgG column wasconcentrated and separated by SDS-PAGE. After transfer to a PVDFmembrane (FIG. 14(B)), the protein band of 45 kDa was excised for directsequencing. The N-terminal sequence of this protein was DVXPVESVX SEQ IDNO:1 (that is, Asp-Val-Xaa-Pro-Val-Glu-Ser-Val-Xaa, wherein Xaarepresents any of the 20 amino acids that occur naturally in humans).The 45 kDa protein represents a unique protein, since no homologousproteins in Genbank were identified.

All patents and references mentioned hereinabove are hereby incorporatedby reference in their entirety. While the foregoing invention has beendescribed in some detail for purposes of clarity and understanding, itwill be appreciated by one skilled in the art from a reading of thedisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention in the appended claims.

1 1 9 PRT Sus scrofa domestica VARIANT Xaa may be any of the 20 aminoacids which occur naturally in mammals. 1 Asp Val Xaa Pro Val Glu SerVal Xaa 1 5

What is claimed is:
 1. A method for detecting the presence of axenoreactive antibody in a sample, comprising the steps of: (a)contacting said sample with a purified porcine red blood cell antigen,said antigen not including an α-Gal epitope, and said antigen comprisingthe amino acid sequence Asp-Val-Xaa-Pro-Val-Glu-Ser-Val-Xaa (SEQ IDNO:1), under conditions permitting said xenoreactive antibody, ifpresent in said sample, to bind to said antigen to form anantigen-antibody complex; and (b) detecting the presence of saidantigen-antibody complex.
 2. A kit for detecting the presence of axenoreactive antibody in a solution, comprising: (a) a container; (b) apurified porcine red blood cell antigen, said antigen not including anα-Gal epitope, and said antigen comprising the amino acid sequenceAsp-Val-Xaa-Pro-Val-Glu-Ser-Val-Xaa (SEQ ID NO:1), which binds to saidxenoreactive antibody to form an antigen-antibody complex; and (c) areagent capable of detecting said antigen-antibody complex.